Through the action of its membrane bound type I receptor, TGF-beta elicits a wide range of cellular responses that regulate cell proliferation, differentiation and apoptosis. Many of these signaling responses are mediated by Smad proteins. As such, controlling Smad activity is crucial for proper signaling by TGF-beta and its related factors. We found that TGF-beta induces phosphorylation at three sites in the Smad3 linker region in addition to the two C-terminal residues, and GSK3 is responsible for phosphorylation at one of these sites, namely Ser204. Alanine substitution at Ser204 and/or the neighboring Ser208, the priming site for GSK3 in vivo activity, strengthened the affinity of Smad3 to CBP, suggesting that the linker phosphorylation may be part of a negative feedback loop that modulates Smad3 transcriptional activity. Thus, our findings reveal a novel aspect of Smad3 signaling mechanism that controls the final amplitude of cellular responses to TGF-beta. In addition to phosphorylation, Smad proteins are also subjected to ubiquitin-mediated degradation. The HECT domain-containing ubiquitin ligase Smurf1 and Smurf2 have the ability to interact directly with Smad1 and Smad5 of the BMP pathway and Smad2 and Smad3 of TGF-beta pathway to regulate their activities. To address the physiological significance of Smurfs in TGF-beta signaling, we have generated mice lacking either Smurf1 or Smurf2.Although Smads are involved in most actions of the TGF-beta superfamily, activated TGF-beta receptors also transduce signals through other intracellular signaling pathways, especially those mediated by MAP kinases. The second area of research of my group focuses on the specific mechanism by which TGF-beta receptors activate MAP kinases independent of Smads, and the biological significance of this non-Smad dependent pathway in TGF-beta signaling. Toward this goal, we found that TRAF6 is specifically required for the Smad-independent activation of JNK and p38 and its carboxyl TRAF homology domain physically interacts with TGF-beta receptors. TGF-beta induces K63-linked ubiquitination of TRAF6, and promotes association between TRAF6 and TAK1. Our results indicate that TGF-beta activates JNK and p38 through a mechanism similar to that operating in the interleukin-1beta/Toll-like receptor pathway. Currently, we seek to expand this findings to characterize molecular mechanisms of the Smad-independent pathways by identifying proteins that are specifically associated with TGF-beta type I receptor and characterizing their functions. The third direction of my group focuses on the effect of aberrant Smad signaling in tumorigenesis. We have generated different lines of transgenic mice carrying either wild type, or dominant negative or Smad3 under the control of a tetracycline-repressible promoter (tet-off). We crossed these mice to LAP-tTA mice, which allow tetracycline-regulated expression of tetracycline-transactivating protein (tTA) specifically in hepatocytes, to express Smad3 and its variants in liver. We find that elevated Smad3 expression protects liver from chemically induced carcinogenesis due to a heightened hepatic response to apoptotic stimuli. We plan to continue using this model to further explore the role of Smad3 in late stages of liver tumor progression and metastasis. In addition, we are also interested in how Smad signaling converges with other pathways and what kind of roles these cross-talks play in controlling TGF-beta-regulated gene transcription, cell proliferation, differenciation, apoptosis and tumor progression.